Lipoprotein-free Fraction Research Articles

Highly polymorphic apolipoprotein A-IV locus in the baboon.

Apolipoprotein A-IV is found in mesenteric lymph chylomicrons, very low density lipoprotein particles, high density lipoprotein particles, and in the lipoprotein-free fraction of plasma. Apolipoprotein A-IV is polymorphic in a variety of species including human, dog, and horse. Efforts to estimate the impact of apolipoprotein A-IV structural variation on quantitative lipid levels in humans have been limited by the low frequency of the less common alleles. In the baboon, Papio hamadryas anubis, we have found apolipoprotein A-IV to be highly variable at the protein level with five alleles appearing at polymorphic frequency. We have confirmed the autosomal codominant inheritance of these five alleles in pedigreed baboons. The baboon has been shown to be a suitable animal model for the study of atherosclerosis, and the existence of a common, multi-allele apolipoprotein A-IV polymorphism in the baboon may be useful in elucidating the role of apolipoprotein A-IV in lipid metabolism.

Studies on stroke-prone spontaneously hypertensive rats(SHRSP) fed a high-fat and high-cholesterol diet. Changes in serum concentrations and distributions of apolipoproteins A-I, A-IV and E.

The effects of a high-fat and high-cholesterol diet (HFC diet) on serum lipoproteins and apolipoproteins were studied in stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Kyo: Wistar rats (WKY). In particular, the changes in serum concentrations and distributions among lipoprotein fractions of apolipoproteins A-I, A-IV and E (apo A-I, A-IV and E) were investigated in detail. These apolipoproteins are the main protein constituents of high density lipoprotein (HDL) which is considered to be an anti-atherogenic factor and accounts for a large part of the serum lipoproteins in the rat. Serum lipoprotein fractions were isolated by stepwise density-gradient ultracentrifugation. The alterations in lipoprotein fractions and apolipoproteins in lipoprotein fractions were roughly estimated by native gradient polyacrylamide gel electrophoresis and sodium dodecyl sulfate (SDS)-gradient polyacrylamide gel electrophoresis. Next, the concentrations of apo A-I, A-IV and E in serum, serum lipoprotein fractions and serum lipoprotein-free fraction were measured by rocket immunoelectrophoresis according to the method of Laurell as modified by us. Cholesterol was enzymatically determined by a commercially available kit. The results obtained were as follows: 1) A remarkable increase in the very low density lipoprotein (VLDL) and intermediate density lipoprotein (IDL) fractions was observed in WKY and SHRSP. This was associated with a remarkable increase in the cholesterol and apo B contents and with a significant increase in the apo E content. These changes in the VLDL and IDL fractions were more drastic in SHRSP than in WKY, which suggests the promotive effect of hypertension in SHRSP on the production of VLDL and IDL fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma metabolism of apolipoprotein A-IV in humans.

As assessed by molecular sieve chromatography and quantitation by a specific radioimmunoassay, apoA-IV is associated in plasma with the triglyceride-rich lipoproteins, to a high density lipoprotein (HDL) subfraction of smaller size than HDL3, and to the plasma lipoprotein-free fraction (LFF). In this study, the turnover of apoA-IV associated to the triglyceride-rich lipoproteins, HDL and LFF was investigated in vivo in normal volunteers. Human apoA-IV isolated from the thoracic duct lymph chylomicrons was radioiodinated and incubated with plasma withdrawn from normal volunteers after a fatty meal. Radioiodinated apoA-IV-labeled triglyceride-rich lipoproteins, HDL, and LFF were then isolated by chromatography on an AcA 34 column. Shortly after the injection of the radioiodinated apoA-IV-labeled triglyceride-rich lipoproteins, most of the radioactivity could be recovered in the HDL and LFF column fractions. On the other hand, when radioiodinated apoA-IV-labeled HDL or LFF were injected, the radioactivity remained with the originally injected fractions at all times. The residence time in plasma of 125I-labeled apoA-IV, when injected in association with HDL or LFF, was 1.61 and 0.55 days, respectively. When 125I-labeled apoA-IV was injected as a free protein, the radioactivity distributed rapidly among the three plasma pools in proportion to their mass. The overall fractional catabolic rate of apoA-IV in plasma was measured in the three normal subjects and averaged 1.56 pools per day. The mean degradation rate of apoA-IV was 8.69 mg/kg X day. The results are consistent with the conclusions that: apoA-IV is present in human plasma in three distinct metabolic pools; apoA-IV associated with the triglyceride-rich lipoproteins is a precursor to the apoA-IV HDL and LFF pools; apoA-IV in LFF is not a free protein and its turnover rate is faster than that of apoA-IV in HDL; since no transfer of apoA-IV from the HDL or the LFF occurs, these pools may represent a terminal pathway for the catabolism of apoA-IV; and the catabolism of apoA-IV in HDL is dissociated from that of apoA-I although both apoproteins may reside on the same lipoprotein particles.

Human breast-milk factors influencing lipid metabolism by fetal rabbit aorta in organ culture

Effects of human or rabbit milk on cholesterol content, incorporation of [14C]oleate, and DNA synthesis were investigated in organ cultures of aorta from fetal and suckling rabbits. Human skim milk (50 mL/L) in organ culture decreased content (mumol/g protein) of aortic total cholesterol (control: 162.9 +/- 24.6 milk: 117.6 +/- 4.9) with significant decrease in cholesteryl esters (control: 16.5 +/- 5.1, milk: 2.3 +/- 0.5). The effect was observed in lipoprotein (total cholesterol: 120.2 +/- 8.8) and lipoprotein-free (total cholesterol: 85.4 +/- 5.1) fractions with molecular weight greater than 100,000. The human milk fraction with molecular weight greater than 100,000 was also the most active in promoting [3H]thymidine incorporation into DNA in fetal aorta. Although milk stimulated the incorporation of [14C] oleate into triglycerides (control: 38.8 +/- 2.5%, milk: 82.1 +/- 4.2%), decreased incorporation to phospholipids (control: 55.0 +/- 3.0%, milk: 11.3 +/- 1.8%) was observed. These studies suggest that milk contains factors influencing aortic lipid metabolism during development.

Structural modifications of rat serum high density lipoprotein by pancreatic phospholipase A2.

An important and unusual aspect of the high density lipoprotein (HDL) in the rat is its tendency to undergo marked alterations in structure in response to physiological perturbations. In this study, the role of the surface lipids for maintenance of HDL integrity were investigated. Hydrolysis by pancreatic phospholipase A2 of the phospholipids of rat HDL in the presence of the d greater than 1.21 g/ml fraction of rat serum results in an increase in the particle diameter and an uptake of apo-E and apo A-IV from the lipoprotein-free fraction; augmentation of the albumin concentration in the incubation mixture intensified the observed changes, probably due to enhancement of the compositional changes brought about by phospholipase treatment. Phospholipase A2, treatment of the d less than 1.21 g/ml fraction of rat serum produces only minor changes in the properties of the isolated HDL. These data suggest that changes in apoprotein content reflect an uptake of A-IV and E by the rat HDL, rather than a net loss of apo A-I. Likewise, titration of the action of pancreatic phospholipase A2 on HDL apoprotein composition showed that initially a modest increase in apo A-IV content occurred, but with more extensive phospholipolysis there was a considerably greater increase in the apo-E content of the particle. The data suggest that hydrolysis of phospholipids such as occurs physiologically through the action of lecithin:cholesterol acyl transferase and hepatic lipase may alter the HDL structure independently from changes effected in the neutral lipid core.

Plasma lipoprotein abnormalities in heterozygotes for familial lecithin:cholesterol acyltransferase deficiency

Measurement of plasma lecithin:cholesterol acyltransferase (LCAT) activity was used to segregate unaffected family members (n = 8) from heterozygotes (n = 8) and homozygotes (n = 2) in a large LCAT-deficient kindred. The activity was absent in the homozygotes and was decreased to 50% of normal in the heterozygotes. Endogenous cholesterol esterification rate measurements did not differentiate the heterozygotes from the unaffected family members or normal subjects. The heterozygotes had significantly higher fasting plasma triglycerides, apo B, and lower HDL-cholesterol and apo AI than the unaffected family members. The HDL of the heterozygotes had the same mass of free cholesterol and triglyceride, but the mass of cholesteryl ester was reduced by 47%. The differences were not related to abnormal postheparin lipolytic activities. However, cholesteryl ester transfer activity in the lipoprotein-free (d > 1.21 bottom) fraction of plasma was significantly ( P < .05) decreased in the heterozygotes when compared to unaffected members. We conclude that the low LCAT activity is the likely cause of the qualitative and quantitative differences in the plasma lipoproteins of the heterozygotes in this family with LCAT deficiency. However, the low HDL and apo A-I levels are not associated with either a family or personal history of premature atherosclerosis.

Effect of lecithin:cholesterol acyltransferase on distribution of apolipoprotein A-IV among lipoproteins of human plasma.

The effect of cholesterol esterification on the distribution of apoA-IV in human plasma was investigated. Human plasma was incubated in the presence or absence of the lecithin:cholesterol acyltransferase (LCAT) inhibitor 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) and immediately fractionated by 6% agarose column chromatography. Fractions were monitored for apoA-IV, apoE, and apoA-I by radioimmunoassay (RIA). Incubation resulted in an elevated plasma concentration of cholesteryl ester and in an altered distribution of apoA-IV. After incubation apoA-IV eluted in the ordinarily apoA-IV-poor fractions of plasma that contain small VLDL particles, LDL, and HDL2. Inclusion of DTNB during the incubation resulted in some enlargement of HDL; however, both cholesterol esterification and lipoprotein binding of apoA-IV were inhibited. Addition of DTNB to plasma after incubation and prior to gel filtration had no effect on the apoA-IV distribution when the lipoproteins were immediately fractionated. Fasting plasma apoE was distributed in two or three peaks; in some plasmas there was a small peak that eluted with the column void volume, and, in all plasmas, there were larger peaks that eluted with the VLDL-LDL region and HDL2. Incubation resulted in displacement of HDL apoE to larger lipoproteins and this effect was observed in the presence or absence of DTNB. ApoA-I was distributed in a single broad peak that eluted in the region of HDL and the gel-filtered distribution was unaffected by incubation either in the presence or absence of DTNB. Incubation of plasma that was previously heated to 56 degrees C to inactivate LCAT resulted in no additional movement of apoA-IV onto lipoproteins, unless purified LCAT was present during incubation. The addition of heat-inactivated LCAT to the incubation, had no effect on movement of apoA-IV. These data suggest that human apoA-IV redistribution from the lipoprotein-free fraction to lipoprotein particles appears to be dependent on LCAT action. The mechanism responsible for the increased binding of apoA-IV to the surface of lipoproteins when LCAT acts may involve the generation of "gaps" in the lipoprotein surface due to the consumption of substrate from the surface and additional enlargement of the core. ApoA-IV may bind to these "gaps," where the packing density of the phospholipid head groups is reduced.

Characterization of serum apolipoprotein patterns in rats during suckling and post-weaning periods

Changes of serum apolipoprotein patterns during the suckling and post-weaning periods were studied in rats. Concentrations of apolipoprotein A-IV and the high-molecular-weight form of apolipoprotein B were markedly high during the early suckling periods and decreased at weaning. Secretion of apolipoprotein A-IV into the mesenteric lymph in 2-week-old rats was as high as that in adult rats into which the high-fat diet was infused constantly. Apolipoprotein A-IV was found both in high-density lipoprotein and lipoprotein-free fractions, and the relative distribution in the latter decreased developmentally. The concentration of apolipoprotein A-I was low for 1 week after birth, after which it increased to the adult level. The apolipoprotein E level during the suckling and post-weaning periods was similar to or above that of adult rats. The newly formed apolipoprotein B in very-low-density lipoproteins secreted by the isolated liver and by the primary culture hepatocytes of suckling rats was predominantly a high-molecular-weight form. Overnight fasting and early weaning caused a remarkable alteration of the serum apolipoprotein profile. It therefore appears that frequent ingestion of dam's milk as well as ontogenic development are relevant to the serum apolipoprotein patterns characteristic for suckling rats.

Mechanisms of enhanced cholesteryl ester transfer from high density lipoproteins to apolipoprotein B-containing lipoproteins during alimentary lipemia.

In vitro lipoprotein lipase enhances the cholesteryl ester transfer protein (CETP)-mediated transfer of cholesteryl esters from high density lipoproteins (HDL) to very low density lipoproteins as a result of lipolysis-induced alterations in lipoprotein lipids that lead to increased binding of CETP. To determine if there are similar changes during alimentary lipemia, we measured the transfer of cholesteryl esters from HDL to apo B-containing lipoproteins in incubated fasting and postprandial plasma. In seven normolipidemic subjects there was 2-3-fold stimulation of cholesteryl ester transfer in alimentary lipemic plasma. Cholesteryl ester transfer was stimulated when either the d less than 1.063-or d greater than 1.063-g/ml fraction of lipemic plasma was recombined with its complementary fraction of fasting plasma. To determine the distribution of CETP, plasma was fractionated by agarose chromatography and CETP activity was measured in column fractions in a standardized assay. In fasting plasma, most of the CETP was in smaller HDL, and a variable fraction was nonlipoprotein bound. During lipemia there was increased binding of CETP to larger phospholipid-enriched HDL and in two subjects an increase in CETP in apo B-containing lipoproteins. The total CETP activity of fractions of lipemic plasma was increased 1.1-1.7-fold compared with fasting plasma. Lipemic CETP activity was also increased when measured in lipoprotein-free fractions after dissociation of CETP from the lipoproteins. When purified CETP was incubated with phospholipid-enriched HDL isolated from alimentary lipemic or phospholipid vesicle-treated plasma, there was increased binding of CETP to the phospholipid-enriched HDL compared with fasting HDL, with a parallel stimulation in CETP activity. Thus, the pronounced stimulation of cholesteryl ester transfer during alimentary lipemia is due to (a) an increased mass of triglyceride-rich acceptor lipoproteins, (b) a redistribution of CETP, especially increased binding to larger phospholipid-enriched HDL, and (c) an increase in total activity of CETP, perhaps due to an increased CETP mass.

Fractionation of chylomicrons by heparin-sepharose chromatography. Characterization of two heparin-binding proteins.

Rat lymph chylomicrons were separated into two fractions using heparin-Sepharose chromatography: a major fraction which elutes from the column with the void volume at 0.05 M NaCl, and a smaller fraction which binds to the column at 0.05 M NaCl and elutes at 0.3 M NaCl. These two fractions differ in mean particle size, and lipid and protein compositions. Both fractions share apolipoproteins B, A-IV, E, A-I, and C, but the fraction which binds to heparin-Sepharose contains two additional proteins: protein I (Mr = 6.0 X 10(4)), and protein II (Mr = 8.0 X 10(4)). Both proteins are also present in the lipoprotein-free fraction of rat serum. Proteins I and II bind to heparin-Sepharose, and are highly amphiphilic: they bind with high affinity to phospholipid surfaces and form stable monolayers at the air-water interface. The molecular weight, amino acid composition, heparin binding, and amphiphilicity of protein I resemble that of beta 2-glycoprotein I; in addition, protein I from rat lymph chylomicrons cross-reacts with rabbit antiserum to human beta 2-glycoprotein I, suggesting that these two proteins are homologous. Protein II appears to be a previously undescribed protein. The possible functions of these two proteins are discussed.

Apolipoprotein changes associated with the plasma lipid-regulating activity of gemfibrozil in cholesterol-fed rats.

Gemfibrozil (Lopid) is a new plasma lipid-regulating drug that decreases very low and low density lipoprotein (VLD/LDL) and increases high density lipoprotein (HDL) concentrations in man. The present experiments tested the effects of gemfibrozil on plasma lipoproteins and apolipoproteins in rats fed high fat/high cholesterol diets. Compared to chow-fed rats, cholesterol feeding for 2 weeks (20% olive oil/2% cholesterol) produced the expected increases in VLDL and intermediate density lipoprotein (IDL) while lowering plasma HDL. This was documented by using three methods of lipoprotein isolation: sequential ultracentrifugation, density gradient ultracentrifugation, and agarose gel filtration. Gemfibrozil gavaged at 50 mg/kg per day for 2 weeks during cholesterol feeding prevented these changes such that lipoprotein patterns were similar to those in chow-fed animals. Whole plasma apoE and apoA-I concentrations were decreased and apoB increased due to cholesterol feeding as determined by electroimmunoassay, but again gemfibrozil treatment prevented these diet-induced alterations. Gradient polyacrylamide gel electrophoresis patterns of the total d less than 1.21 g/ml lipoprotein fractions reflected the changes in apolipoprotein concentrations and further demonstrated a greater increase of apoBl compared to apoBh in cholesterol-fed rats. Gemfibrozil lowered the concentration of both apoB variants and prevented the shift of apoE from HDL to lower density lipoproteins. Changes in the distribution of apoE were confirmed using agarose gel column chromatography followed by electroimmunoassay. These methods also revealed a shift of apoA-IV from HDL to the d greater than 1.21 g/ml, lipoprotein-free fraction with gemfibrozil treatment when blood was taken from fasted or postabsorptive animals. Since it was also noted that in chow-fed rats more apoA-IV was present in the d greater than 1.21 g/ml fraction in the postabsorptive or fed state compared to fasted animals, it could be postulated that the shift of apoA-IV into this fraction in gemfibrozil-treated rats is related to an accelerated clearance of chylomicrons. It is concluded that gemfibrozil largely prevents the accumulation of abnormal lipoproteins in this model of dyslipoproteinemia, and that apoE may play a critical role in this normalization process.

Role of lipid transfers in the formation of a subpopulation of small high density lipoproteins.

The effect of lipid transfers on the structure and composition of high density lipoproteins (HDL) has been studied in vitro in incubations that contained the lipoprotein-free fraction of human plasma as a source of lipid transfer protein. These incubations did not contain lecithin:cholesterol acyltransferase activity and were not supplemented with lipoprotein lipase. Incubations were performed at 37 degrees C for 6 hr in both the presence and absence of either added very low density lipoproteins (VLDL) or the artificial triglyceride emulsion, Intralipid. Incubation in the absence of added VLDL or Intralipid had little or no effect on the HDL. By contrast, incubation in the presence of either VLDL or Intralipid resulted in marked changes in the HDL. The effect of incubation with VLDL was qualitatively similar to that of Intralipid; both resulted in obvious transfers of lipid and changes in the density, particle size, and composition of HDL. Incubation of the plasma fraction of density 1.006-1.21 g/ml, total HDL, or HDL3 with either VLDL or Intralipid resulted in the following: 1) a depletion of the cholesteryl ester and free cholesterol content and an increase in the triglyceride content of both HDL2 and HDL3; 2) a decrease in density and an increase in particle size of the HDL3 to form a population of HDL2-like particles; and 3) the formation of a discrete population of very small lipoproteins with a density greater than that of the parent HDL3. The newly formed lipoproteins had a mean particle radius of 3.7-3.8 nm and consisted mainly of protein, predominantly apolipoprotein A-I and phospholipid.

Changes in lipid and apolipoprotein composition of pig lipoproteins facilitated by rabbit lipid transfer protein

A lipid transfer protein has been purified from the lipoprotein-free fraction of rabbit plasma. Rabbit lipid transfer protein, which was purified 600–700-fold with a 4% recovery, has an apparent M r. of 68000 and facilitates the transfer of isotopically labelled cholesterol ester, triacylglycerol and phosphatidylcholine between low-density lipoprotein (LDL) and high-density lipoprotein (HDL). Rabbit lipid transfer protein, which appears to be very similar to the cholesteryl ester exchange protein previously purified from human plasma, was incubated with pig plasma at 37° C for up to 6 h. Analysis of very-low-density lipoprotein (VLDL, d < 1.006 g/ml), LDL ( d 1.019−1.063 g/ml) and HDL ( d 1.090−1.21 g/ml) after incubation showed that lipid transfer protein had a marked effect on the composition of the lipoprotein classes. The VLDL became enriched with cholesteryl ester and depleted of triacylglycerol. The LDL and HDL became enriched with triacylglycerol. In addition to these changes in lipid composition there were also changes in apolipoprotein composition. The most prominent change in apolipoprotein distribution was a marked increase in the apolipoprotein E content of LDL which was observed only after incubation in the presence of lipid transfer protein.

Distribution of apolipoprotein A-IV between the lipoprotein and the lipoprotein-free fractions of rat plasma: possible role of lecithin:cholesterol acyltransferase.

Plasma samples were incubated under various conditions to study the effect of in vitro incubation on apolipoprotein A-IV distribution between the lipoprotein and lipoprotein-free fractions. When plasma was fractionated immediately after bleeding, apolipoprotein A-IV was present in equal concentrations in the lipoprotein and lipoprotein-free fractions. After a 4-hr, 37 degrees C incubation, greater than 90% of total plasma apolipoprotein A-IV was present in the lipoprotein fraction and the percentage of plasma cholesterol present as cholesteryl ester increased from 58% to 74%. When plasma was incubated for 4 hr at 37 degrees C in the presence of 1.5 mM 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), greater than 90% of total plasma apoA-IV was present in the lipoprotein-free fraction, whereas plasma cholesteryl ester concentration did not change. Incubating heat-inactivated plasma for 4 hr also resulted in the redistribution of apolipoprotein A-IV from the lipoprotein fraction to the lipoprotein-free fraction, concurrent with no change in cholesterol esterification. When heat-inactivated plasma was incubated in the presence of a purified lecithin:cholesterol acyltransferase preparation, cholesterol esterification was restored and apolipoprotein A-IV was redistributed from the lipoprotein-free fraction to the lipoprotein fraction in such a manner that greater than 90% was present in the lipoprotein fraction. No changes in apolipoprotein A-I and apolipoprotein E distributions were found under any of the above conditions. Thus, the in vitro plasma incubations show that apolipoprotein A-IV can move bidirectionally between lipoprotein and lipoprotein-free fractions; the direction of this movement depends on the condition of the incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

The response of apolipoprotein A-IV to cholesterol feeding in rats

The effect of feeding high-fat and high-fat, high-cholesterol diets on the concentrations and distributions of apolipoprotein A-IV and A-I was studied. Rats were fed the diets as a single meal or for a period of 4 weeks. Feeding a single meal increased serum apolipoprotein A-IV concentrations above prefeeding values, regardless of the dietary composition. After a single meal was fed, serum apolipoprotein A-IV concentrations were lowest in the rats given the control diet and significantly higher in those fed the olive oil or olive oil /cholesterol diets. Feeding increased the apolipoprotein A-IV concentration in the lipoprotein-free fraction, regardless of the diet given. The increase in the lipoprotein-free apolipoprotein A-IV concentration was greater than control when olive oil was added to the diet and even greater when olive oil and cholesterol were added to the diet. In the olive oil/cholesterol group, not only was the lipoprotein-free apolipoprotein A-IV concentration greater than those of the control and olive oil groups but the HOL apolipoprotein A-IV concentration was also less than those of both the control and the olive oil groups. This finding shows that the presence of cholesterol in the diet specifically influenced the apolipoprotein A-IV distribution. When the rats were fed the high-fat, high-cholesterol diets for 4 weeks, the decrease in HDL apolipoprotein A-IV concentration and increase in lipoprotein-free apolipoprotein A-IV concentration was also observed. The changes were confirmed by agarose gel column Chromatography. In contrast to apolipoprotein A-IV, the serum concentrations and distributions of apolipoprotein A-I remained relatively constant with all experimental manipulations; however, with long-term feeding, serum apolipoprotein A-I concentration was significantly decreased in the olive oil /cholesterol-fed rats. We conclude that the metabolism of apolipoprotein A-IV may be related to cholesterol metabolism.

Changes in the concentrations and distributions of apolipoproteins of the aging rat

The hyperlipidemia associated with aging was characterized in the rat by comparing the plasma lipid, lipoprotein, and apolipoprotein profiles of adult (12 weeks old) and old (96 weeks old) male rats. Compared with those of the adult rats, the VLDL concentrations of the old rats were reduced, but IDL, LDL, and HDL concentrations were elevated. Despite a reduced VLDL concentration, concentrations of triglycerides in the plasma of the old rats were elevated. This phenomenon was attributed to an enrichment of triglyceride in the other lipoprotein fractions. In the old rats, hypercholesterolemia was the result of elevated IDL- and HDL-cholesterol whereas elevated plasma concentrations of apolipoproteins B and E were attributed to elevated LDL and HDL concentrations, respectively. Although concentrations of apolipoproteins A-I and A-IV did not change significantly in the plasma of the old rats, the distribution pattern of the apoA-IV was altered dramatically. Compared with the adult rats, a shift of apoA-IV in the HDL to the "lipoprotein-free" fraction was observed in the old rats, as measured by agarose gel chromatography. The data demonstrate that the hyperlipidemia in the old rats is associated with selective changes in the apolipoprotein profile.

Neutralization of bacteria- and endotoxin-induced hypotension by lipoprotein-free human serum.

Normal human serum and a fraction rich in lipoprotein, Cohn fraction IV1, have been shown in previous studies to detoxify native endotoxin by decreasing lethality for mice, fever in rabbits, and by the alteration of the characteristic endotoxin-anti-endotoxin precipitin pattern in gels. These studies are extended herein and document the ability of normal human serum and fraction IV1 to neutralize the induction of hypotension in rabbits by viable gram-negative bacilli. Further fractionation of serum, using an ultracentrifugal flotation method for producing lipoprotein-free human serum and purified high-density lipoproteins, revealed the lipoprotein-free fraction to be capable of inhibiting endotoxin hypotensive activity and to alter diffusion of endotoxin in gels. On the other hand, the purified high-density lipoproteins failed to negate either activity.

Apoprotein C metabolism in man.

This chapter focuses on apoprotein C metabolism in man. The C apoprotein (apoC) group is closely linked to triglyceride metabolism, and the identification of one peptide, later called apoC-II, as the activator of lipoprotein lipase represented the first demonstration of a specific function for an apoprotein. Human lipoproteins have generally been preferred for in vitro experiments, and probably more has been learned from the metabolic investigations carried out in humans than in experimental animals. In the presence of plasma, it is evident that apoC species exchange between VLDL and HDL. Although apoC has also been found in lipoprotein—free fractions when released from VLDL in the absence of serum or HDL, this does not mean that they will exist in this form under physiological conditions. Furthermore, the very marked affinity of apoC for phospholipids at physiological temperatures would suggest that, even if liberated as pure apoproteins, they would mostly bind to lipoproteins where phospholipids are present in an ordered surface structure such as HDL.

Evidence for a lipoprotein carrier in human plasma catalyzing sterol efflux from cultured fibroblasts and its relationship to lecithin:cholesterol acyltransferase.

Immunoaffinity chromatography has been used to study the determinants of sterol efflux and net transport from cultured fibroblasts to human plasma medium. Sterol efflux was highly (approximately 80%) dependent upon a minor lipoprotein fraction containing apolipoprotein A-I unassociated with other apolipoproteins. The remaining activity was associated with the lipoprotein-free fraction of plasma and could be replaced by apoprotein-free albumin. Efflux was independent of lecithin:cholesterol acyltransferase (EC 2.3.1.43) activity. Net transport (i.e., the excess of efflux over influx) was completely inhibited by inhibition of lecithin:cholesterol acyltransferase or its removal by affinity chromatography on immobilized antibodies to apolipoprotein A-I or D (components of the transfer complex in human plasma). In uninhibited plasma, efflux and net transport rates had similar kinetics, suggesting that these were linked functions and that net transport was initiated by a carrier-dependent efflux step that, in the absence of lecithin:cholesterol acyltransferase activity, was associated with an equivalent influx of free sterol to the cells and that, in the presence of lecithin:cholesterol acyltransferase, was associated with esterification and transfer protein activity. The cholesterol carrier lipoprotein function (approximately 5% of plasma apolipoprotein A-I) appears to be the first step of lecithin:cholesterol acyltransferase-linked sterol transport from cells.

The redistribution and metabolism of iodinated apolipoprotein A-IV in rats

The metabolic fate of 125I-labeled apolipoprotein A-IV has been studied in rats. This was achieved by incorporating labeled apolipoprotein A-IV into lymph chylomicrons and plasma high density lipoprotein by in vitro exchange or by association, in vivo of apolipoprotein A-IV with plasma chylomicrons and high density lipoprotein following injection of 125I-labelled apolipoprotein A-IV into fat-fed rats. After separating lipoprotein-bound from unbound apolipoprotein A-IV, the interrelationship of apolipoprotein A-IV between lipoproteim; was studied by administering 125I-labeled apolipoprotein A-IV/lipoproteins into rats and bleeding at various times thereafter. Apolipoprotein A-IV was rapidly removed from chylomicrons and transferred into high density lipoprotein and lipoprotein-free fractions. The time course of transfer, measured both as absolute radioactivity and as specific radioactivity was consistent with a precursorproduct relationship between apolipoprotein A-IV in the lipoprotein-free fraction and high density lipoprotein, suggesting that the rapid transfer of apolipoprotein A-IV from chylomicrons into the lipoprotein-free form precedes transfer into the high density lipoprotein fraction. Since it could be demonstrated by electroimmunoassay that approximately half the total apolipoprotein A-IV in rat plasma was not associated with lipoproteins, it is concluded that a pool of lipoprotein-free apolipoprotein A-IV exists which may have an important physiological role in the lipoprotein transport system.